Dual Lentiviral Transduction To Generate Mutant Neural Stem Cells For Glioma Research

Gliomas are the most prevalent primary brain tumors in adults, and murine modeling suggests that they may arise from oncogenic mutations in NSCs or early differentiating progenitors. Orthotopic transplantation models derived from mutant NSCs thus have the potential to yield valuable preclinical information about glioma pathogenesis, outcomes, and treatment response. These models require the introduction of an oncogenic mutation into NSCs, preferentially coupled with reporters to aid in the determination of transfection/transduction efficiency and to monitor tumor growth in vivo. This protocol outlines a dual transduction-based methodology for generating mutant NSCs suitable for in vivo tumor monitoring. In this technique, isolated NSCs are first transduced with lentiviruses that contain a gene of interest (oncogenic mutation or green fluorescent protein [GFP] control) in addition to a blasticidin-resistance element. Following blasticidin selection, cells are secondarily transduced with a bicistronic lentiviral vector expressing luciferase and red fluorescent protein (RFP), as well as a puromycin-resistance element. After puromycin selection, cells can be expanded for proliferation assays, orthotopic transplantation, or other downstream applications. This dual transduction approach allows for estimation of transduction efficiency at both infection stages as well as monitoring in vivo tumor establishment, growth, and response to therapy using noninvasive bioluminescent imaging (BLI). This system represents a useful tool for glioma research, merging the biological relevance of adult neural stem cell-derived tumor modeling with the practical benefit of real-time imaging capability, ultimately enhancing the understanding of glioma biology and the advancement of new therapeutic approaches.